Method for Identifying and Selecting Cardiomyocytes

ABSTRACT

The present invention relates to new and/or improved methods of identification and selection of cardiomyocytes from human embryonic stem (hES) cells. The method further comprises isolating the selected cardiomyocyte population. There is also provided method for the screening for cardiovascular compounds comprising subjecting the said cardiomyocyte population to test compound/s, and observing and/or interpreting a response of the cardiomyocytes to the test compound.

FIELD OF THE INVENTION

The present invention relates to the identification and isolation ofcardiomyocytes from human embryonic stem (hES) cells.

BACKGROUND OF THE INVENTION

Cardiovascular diseases remain the leading cause of mortality andmorbidity world wide. Since adult cardiomyocytes do not regenerate, thedeath of these cells compromises the myocardial contractile function.For instance when the coronary vessel is occluded by a thrombus and thesurrounding cardiomyocytes cannot be supplied with necessary energysources from other coronary vessels. The loss of functionalcardiomyocytes may lead to chronic heart failure. A potential route ofrestoring normal heart function is replacement of injured and deadcardiomyocytes by new functional cardiomyocytes.

The success of regenerative cardiac medicine depends on the availabilityof cardiomyocytes in sufficient numbers for the transplantation of thecardiac tissue. Cardiomyocytes have the potential to restore heartfunction after myocardial infarction or heart failure and humanembryonic stem (hES) cells are potential source of transplantablecardiomyocytes (Siu et al, 2007).

A limitation in the study of cardiomyocytes has been the inability toidentify these cells prospectively. The current protocols designed todirect the differentiation of human embryonic stem cells in vitrotowards cells of the cardiomyocyte lineage produce a heterogeneouspopulation of cells of various identity and developmental stage. For thepurpose of producing cell therapies or diagnostic cell products, pure orrelatively pure cardiomyocyte populations are desired. Nearly purepopulations of cardiomyocytes have been generated from mouse embryonicstem cells using a method requiring prior genetic transformation of thestem cells. Genetic transformation of stem cells is time consuming andmay preclude the enriched cell population from use in the clinic. Itwould therefore be an advantage to have a method capable of isolating apopulation of differentiated cells enriched for cardiomyocytes fromwild-type stem cells. Furthermore, it would be an advantage if a largeproportion of the enriched cardiomyocytes were viable and capable ofproliferation, allowing the enriched population to expand in culture fora number of population doublings.

SUMMARY OF THE INVENTION

The present invention addresses the problems above and in particularprovides new and improved method of identification and isolation ofcardiomyocytes from differentiated embryonic stem (ES) cells.

According to a first aspect, the present invention provides a method ofidentifying and selecting a cardiomyocyte population from aheterogeneous population of differentiated stem cells, comprisingcontacting the heterogeneous cell population with at least one agentthat specifically binds to at least one cardiomyocyte marker andselecting the bound cells as cardiomyocytes.

The method further comprises isolating the selected cardiomyocytepopulation. There is also provided a method of propagating the selectedcardiomyocyte population in culture. In particular, the at least onecardiomyocyte marker is selected from a group consisting of CD166(ALCAM), VEGF receptor Flk1, N-cadherin, CD133 and CD117 (C-kit). Morein particular the at least one cardiomyocyte marker is CD166 (ALCAM).The at least one cardiomyocyte marker may be a fetal marker. Theidentified cells may comprise at least 50% cardiomyocytes. In particularthe identified cardiomyocytes may have a fetal phenotype. For examplecardiomyocytes may be capable of proliferating in culture. In particularat least 25% of the identified cardiomyocytes may be in S phase of thecell cycle. More in particular the identified cardiomyocytes are capableof rhythmic contractions and/or forming electrically coupled cellclusters. As a non-limitative example, the stem cells may be selectedfrom a group consisting of embryonic stem (ES) cell, pluripotent stemcells, hematopoietic stem cells, totipotent stem cells, mesenchymal stemcells, neural stem cells and adult stem cells. In particular the stemcells may be human ES cells.

According to another aspect, the invention provides a cardiomyocytepopulation having the characteristics as herein defined. In particular,there is provided a cardiomyocyte population identified and/or isolatedby the method according to the present invention. There is also provideda cardiomyocyte population isolated according to the method of thepresent invention.

According to yet another aspect, the invention provides a model forstudy of human cardiomyocytes in culture, comprising the cardiomyocytepopulation. The invention further provides a kit for cardiotoxic testingcomprising the cardiomyocyte population.

Another aspect of the invention includes a method of preventing,repairing and/or treating at least one cardiac disorder in a subject,the said method comprising transplanting the isolated cardiomyocytepopulation. The cardiac disorder may be selected from a group consistingof myocardial infarction, cardiomyopathy, congestive heart failure,ventricular septal defect, atria septal defect, congenital heart defectand ventricular aneurysm.

According to a further aspect, the invention provides a model fortesting suitability of cardiomyocytes for cardiac transplantation, saidmodel comprising:

-   -   A non-human animal having a measurable parameter of cardiac        function wherein the said animal is capable of receiving an        isolated cardiomyocyte population; and    -   a means to determine cardiac function of the animal before and        after transplantation of the isolated cardiomyocyte population.        In particular the model may be an immunodeficient animal created        as a model of cardiac muscle degeneration following infarct that        is used as a universal acceptor of the isolated cardiomyocyte        population. More in particular the animal model may be murine,        ovine, bovine, porcine or a non-human primate. More in        particular the parameter of cardiac function may be contractile        function.

According to yet another aspect the invention provides a method ofscreening for cardiovascular compounds. In particular the method maycomprise subjecting the said cardiomyocyte population to at least onetest compound, and observing a cardiac specific response of thecardiomyocytes to at least one test compound. In particular the cardiacspecific response may comprise alteration of Q-T wave.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 represents the expression of the cardiac transcription factorNkx2.5 analysed by immunofluorescence following culturing of humanembryonic stem cells for 14 days, under conditions which promotecardiomyocyte differentiation. The nuclei are counterstained with DAPI(blue), the area shown by arrows.

FIG. 1A represents the co-localization of Nkx2.5 (green) with thecardiac marker αMHC (red). The black and white view of FIG. 1Arepresents the co-localization of Nkx2.5 (dark white) with the cardiacmarker αMHC (light grey)

FIG. 1B represents the co-localization of Nkx2.5 (red) with the cardiacmarker MLC2a (green). The black and white view of FIG. 1B represents theco-localization of Nkx2.5 (light grey) with the cardiac marker MLC2a(dark white).

FIG. 1C represents the co-localization of Nkx2.5 (red) with the cardiacmarker alpha-actinin (green). The black and white view of FIG. 1Crepresents the co-localization of Nkx2.5 (light grey) with the cardiacmarker alpha-actinin (dark white).

FIG. 1D represents the co-localization of Nkx2.5 (red) with the cardiacmarker tropomyosin (green). The black and white view of FIG. 1Drepresents the co-localization of Nkx2.5 (light grey) with the cardiacmarker tropomyosin (dark white).

FIG. 1E represents the co-localization of Nkx2.5 (red) with the cardiacmarker MLC2v (green). The black and white view of FIG. 1E represents theco-localization of Nkx2.5 (light grey) with the cardiac marker MLC2v(dark white).

FIG. 2 represents the expression of the cardiac transcription factorNkx2.5 (green) analysed by immunofluorescence following culturing ofhuman embryonic stem cells for 14 days under conditions which promotecardiomyocyte differentiation. The nuclei are counterstained with DAPI(blue), the area shown by arrows.

FIG. 2A represents the co-localization of Nkx2.5 (green) with thecardiac marker CD166 (red). The black and white view of FIG. 2Arepresents the co-localization of Nkx2.5 (dark white) with the cardiacmarker CD166 (light grey).

FIG. 2B represents the co-localization of Nkx2.5 (green) with thecardiac marker Flk-1 (red). The black and white view of FIG. 2Brepresents the co-localization of Nkx2.5 (dark white) with the cardiacmarker Flk-1 (light grey).

FIG. 2C represents the co-localization of Nkx2.5 (green) with thecardiac marker N-cadherin (red). The black and white view of FIG. 2Crepresents the co-localization of Nkx2.5 (dark white) with the cardiacmarker N-cadherin (light grey).

FIG. 3 represents percentage of surviving adherent cells at 48 hours,following digestion of the embryoid bodies and undifferentiated hES withtrypsin or accumax reagent, and plating of the single cell suspensionson collagen I treated tissue culture dishes.

FIG. 4 represents quantitative PCR analysis of RNA extracted from MACSsorted cells based on expression of CD166. Figure represents theexpression of the cardiac markers Nkx2.5 and αMHC, neural markerNeuroD1, pluripotent cells marker Oct4 and endodermal cells marker AFPon cells isolated based on expression of CD166 enriched forcardiomyocytes.

FIG. 5 represents proliferation of cells in collagen I coated culturedishes, isolated based on expression of CD166.

FIG. 5A represents the sub-confluent layer of surviving cells attachedto the dish after one day in culture.

FIG. 5B represents confluent layer of surviving cells attached to thedish after six days in culture.

FIG. 5C represents the analysis of cells in S phase by BrdU (green)incorporation into the layer of surviving cells attached to the dishafter two days in culture. In black and white view of FIG. 5C representsthe analysis of cells in S phase by BrdU (dark grey) incorporation intothe layer of surviving cells attached to the dish after two days inculture

FIG. 6 represents immunofluorescence analysis of the expression ofNkx2.5 (dark pink) and MLC2a (green) following the sorting of the 14 dayold EBs based on expression of CD166, and plated on collagen I coateddishes in medium containing bovine serum and allowed to grow toconfluence over a period of five days. Nuclei are counterstained withDAPI (blue). In the black and white view the Nk2.5 stained cells appearto be dark white while the DAPI counterstain appear as light grey.

FIG. 6A represents CD166+ cells expressing the cardiomyocyte markerNkx2.5 (dark pink). The black and white view of FIG. 6A representsCD166+ cells expressing the cardiomyocyte marker Nkx2.5 (dark white).

FIG. 6B represents that CD166− cells do not express or express verylittle of the cardiomyocyte marker Nkx2.5 (dark pink). The black andwhite view FIG. 6B represents that CD166− cells do not express orexpress very little of the cardiomyocyte marker Nkx2.5 (dark white)

FIG. 6C represents CD166+ cells expressing the cardiomyocyte markersNkx2.5 (dark pink) and MLC2a (green). The black and white view FIG. 6Crepresents CD166+ cells expressing the cardiomyocyte markers Nkx2.5(dark white) and MLC2a (grey).

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO. 1 refers to Actin forward primer 5′-CAATGTGGCCGAGGACTTTG-3′SEQ ID NO. 2 refers to Actin reverse primer 5′-CATTCTCCTTAGAGAGAAGTG-3′SEQ ID NO. 3 refers to Nkx2.5 forward primer 5′-AGAAGACAGAGGCGGACAAC-3′SEQ ID NO. 4 refers to Nkx2.5 reverse primer 5′-CGCCGCTCCAGTTCACAG-3′SEQ ID NO. 5 refers to αMHC forward primer 5′-ATTGCTGAAACCGAGAATGG-3′SEQ ID NO. 6 refers to αMHC reverse primer 5′-CGCTCCTTGAGGTTGAAAAG-3′SEQ ID NO. 7 refers to NeuroD forward primer 5′-GCCCCAGGGTTATGAGACTA-3′SEQ ID NO. 8 refers to NeuroD reverse primer 5′-GTCCAGCTTGGAGGACCTT-3′SEQ ID NO. 9 refers to Oct4 forward primer 5′-GGCAACCTGGAGAATTTGTT-3′SEQ ID NO. 10 refers to Oct4 reverse primer 5′-GCCGGTTACAGAACCACACT-3′SEQ ID NO. 11 refers to AFP forward primer 5′-GTAGCGCTGCAAACAATGAA-3′SEQ ID NO. 12 refers to AFP reverse primer 5′-TCCAACAGGCCTGAGAAATC-3′

DETAILED DESCRIPTION OF THE INVENTION

Bibliographic references mentioned in the present specification are forconvenience listed in the form of a list of references and added at theend of the examples. The whole content of such bibliographic referencesis herein incorporated by reference.

The present invention provides new and/or improved method ofidentification and isolation of cardiomyocytes from differentiatedembryonic stem (ES) cells.

According to one aspect, the invention provides a method of identifyingand selecting a cardiomyocyte population from a heterogeneous populationof differentiated stem cells, comprising contacting the heterogeneouscell population with at least one agent that specifically binds to atleast one cardiomyocyte marker and selecting cells bound to the saidagent as cardiomyocytes. The heterogeneous population of differentiatedstem cells may be prepared according to the method described in WO2007/030870 (the content of which is herein incorporated by reference).

The method further comprises isolating the selected cardiomyocytepopulation. There is also provided a method of propagating the selectedcardiomyocyte population in culture. In particular, the at least onecardiomyocyte marker is selected from the group consisting of CD166(ALCAM), VEGF receptor Flk1, N-cadherin, CD133 and CD117 (C-kit). Morein particular the at least one cardiomyocyte marker is CD166 (ALCAM).The at least one cardiomyocyte marker may be a fetal marker.

Sorting of cells based on surface marker expression may be accomplishedby using any technology known in the art. For example, sorting of cellsbased on surface marker expression may be accomplished by using FlowAssisted Cell Sorting (FACS) or Automated Magnetic Cell Sorting (MACS)technology. The preparation of the cells for FACS is similar topreparation of cells for MACS except that the secondary antibody isconjugated to a FACS-compatible fluorophore instead of a magneticmicrobead.

At least 50% of the identified, selected and/or isolated cells accordingto the invention may comprise cardiomyocytes. In particular, 55%, 60%,70%, 80% or 90% of the isolated cells may comprise cardiomyocytes. Inparticular the identified, selected and/or isolated cardiomyocytes mayhave a fetal phenotype.

The cardiomyocytes may be capable of proliferating in culture. Inparticular at least 25% of the identified cardiomyocytes may be in Sphase of the cell cycle.

More in particular, the identified cardiomyocytes are capable ofrhythmic contractions and/or forming electrically coupled cell clusters.

As a non-limiting example, the stem cells may be selected from a groupconsisting of embryonic stem (ES) cell, pluripotent stem cells,hematopoitic stem cells, totipotent stem cells, mesenchymal stem cells,neural stem cells and adult stem cells. In particular the stem cells maybe human ES cells. In particular the stem cells may be isolated EScells. For example, the ES cell may be obtained from at least one EScell line recognised the NIH human stem cell registry(http://stemcells.nih.gov/research/registry/defaultpage.asp) accordingto the methods and ethical standards mentioned therein. More inparticular, the hES cell line hES3 from ES Cell International may beused.

“Stem cells” as described herein refers to a stem cell that isundifferentiated prior to culturing and is capable of undergoingdifferentiation. The stem cells may be selected from a group consistingof embryonic stem (ES) cell, pluripotent stem cells, hematopoietic stemcells, totipotent stem cells, mesenchymal stem cells, neural stem cellsand adult stem cells. In particular the stem cell may be human embryonicstem (hES) cells. For example the stem cell may be derived from a cellculture, such as hES cells. The stem call may be derived from anembryonic cell line or embryonic tissue. The embryonic stem cells may becells which have been cultured and maintained in an undifferentiatedstate.

The stem cells suitable for use in the present methods may be derivedfrom a patient's own tissue. This would enhance compatibility ofdifferentiated tissue grafts derived from stem cells with the patient.

Differentiated stem cells may express markers on their cell surface thatmay be indicative of a specific cell type, for example indicative ofcardiomyocytes. The markers may be used to identify and isolate thedifferentiated cardiomyocytes from other differentiated cells andundifferentiated stem cells. “Markers”, as used herein, are polypeptidemolecules that are expressed on a cell of interest. The specific markermay be present only in the cells of interest, or encompass the cells ofinterest, or detectable level of the marker is sufficiently higher inthe cells of interest, compared to other cells, such that the cells ofinterest can be identified, using any of a variety of methods as knownin the art. It will be understood by those of skill in the art thatexpression is a relative term, and the expression will vary from othercell types. For example, a progenitor cell may express a polypeptidethat is not found in the fully differentiated progeny cell. A cell ofinterest may express a polypeptide that is not expressed in surroundingtissues, e.g. the cardiomyocyte cells of fetal phenotype may expressCD166 polypeptides not found in mature cardiomyocytes or on other cellsof a non-cardiomyocyte lineage. This specificity is sufficient forpurposes of cell identification and isolation. Therefore, “fetalmarkers” as used herein refer to a marker on a cell, in particularcardiomyocytes that is indicative of the fetal phenotype of the cells.Fetal phenotype further refers to cells that are capable ofproliferating in culture. Some fetal markers of interest in the presentinvention include CD166 (ALCAM), VEGF receptor Flk1, N-cadherin, CD133,CD117 (C-kit), Nkx2.5, α-MHC, MLC2a, MLC2v, α-actinin and tropomyosin.In particular, fetal markers of interest in the present inventioninclude CD166 (ALCAM), VEGF receptor Flk1, N-cadherin, CD133, CD117(C-kit). More in particular the cardiomyocyte marker may be CD166(ALCAM). These markers are well known in the art, and agents (reagents)for the detection thereof are widely available. In a typical assay fordetection and/or isolation, a heterogeneous population of differentiatedstem cell is contacted with at least one a marker-specific “agent”, anddetecting directly or indirectly the presence of the complex formed. Theterm “agent” as used herein refers to a molecule capable of binding toanother molecule, for example the marker on the cell surface, throughchemical or physical means, wherein the agent and the marker form abinding pair. For example antibodies specific for these cell surfacemarkers are commercially available, or may be produced usingconventional methods as known in the art, therefore the antibodies andmarkers form a binding pair.

Of particular interest is the use of antibodies as affinity reagents.Conveniently, these antibodies are conjugated with a label for use inseparation. Labels include magnetic beads, which allow for directseparation on magnetic assisted cell sorter (MACS), biotin, which can beremoved with avidin or streptavidin bound to a support, fluorochromes,which can be used with a fluorescence activated cell sorter (FACS), orthe like, to allow for ease of separation of the particular cell type.Fluorochromes that find use include phycobiliproteins, e.g.phycoerythrin and allophycocyanins, fluorescein and Texas red.Frequently each antibody is labeled with a different fluorochrome, topermit independent analysis or sorting for each marker. Monoclonalantibodies specific for the markers may be produced in accordance withconventional ways, immunization of a mammalian host, e.g. mouse, rat,guinea pig, cat, dog, etc., fusion of resulting splenocytes with afusion partner for immortalization and screening for antibodies havingthe desired affinity to provide monoclonal antibodies having aparticular specificity. These antibodies can be used for affinitychromatography, ELISA, RIA, and the like. The antibodies may be labelledwith radioisotopes, enzymes, fluorescers, chemiluminescers, or otherlabel which will allow for detection of complex formation between thelabelled antibody and its complementary epitope.

In particular the invention provides methods of preventing, repairingand/or treating at least one cardiac disorder in a subject, the methodcomprising transplanting the cardiomyocyte population in a subject. Thesubject is, in particular, a subject in need of the treatment thereof.The disorder as, used herein, include but are not limited to myocardialinfarction, cardiomyopathy, congestive heart failure, ventricular septaldefect, atria septal defect, congenital heart defect and ventricularaneurysm. In this aspect of the invention, the method includesintroducing a cardiomyocyte population of the invention into cardiactissue of a subject. In particular the isolated cardiomyocyte populationis transplanted into damaged cardiac tissue of the subject. More inparticular the method results in the restoration of cardiac function ina subject. The cardiomyocyte population may resemble a human fetalatrial cell in culture. In particular the cardiomyocyte population mayresemble a human fetal pacemaker cell in culture. More in particular thecardiomyocyte population may comprise plurality of isolatedcardiomyocytes wherein the cardiomyocytes may be coupled. The couplingmay be, for example, through gap junctions and/or adherens junctions,wherein the coupling is electrical. The subject may be a human ornon-human animal.

The present invention also provides at least one cardiomyocytepopulation identified, selected and/or isolated according to the methodof the present invention for use in medicine. In particular, inpreventing, repairing and/or treating at least one cardiac disorder in asubject. There is also provided the use of at least one cardiomyocytepopulation identified, selected and/or isolated according to the methodof the present invention for the preparation of a medicament inpreventing, repairing and/or treating at least one cardiac disorder in asubject.

The present invention also provides a cardiac model for testing theability of the isolated cardiomyocyte population to restorecardiomyocyte function. In order to test the effectiveness oftransplanted cardiomyocyte population in vivo, it is important to have areproducible animal model with a measurable parameter of cardiacfunction. The parameters used should clearly distinguish control andexperimental animals so that the effects of the transplantation can beadequately determined. A host animal, such as, but not limited to, animmunodeficient mouse may be used as a ‘universal acceptor’ ofcardiomyocytes produced by the methods of the present invention.

The myocardial model of the present invention is designed to assess theextent of cardiac repair following transplant of cardiomyocytes into thehost animal. In particular, the host animal may be an immunodeficientanimal created as a model of cardiac muscle degeneration followinginfarct that is used as a universal acceptor of isolated cardiomyocytes.The non-human animal may be any species including but not limited tomurine, ovine, canine, bovine, porcine and any non-human primates.Parameters used to measure cardiac repair in these animals may include,but are not limited to, electrophysiological characteristic of hearttissue or various heart functions. For instance, contractile functionmay be assessed in terms of volume and pressure changes in a heart.Methods of assessing heart function and cardiac tissue characteristicsmay also involve techniques known to person skilled in the art.

The invention further provides cardiomyocytes produced using the methodsof the current invention that may be used for transplantation, celltherapy or gene therapy. In particular the invention provides the use ofcardiomyocytes produced using the methods of the current invention, in acardiac model for testing the ability to restore cardiac function. Morein particular the invention provides the use of cardiomyocytes in acardiac model designed to assess the extent of cardiac repair followingtransplant of cardiomyocytes into a suitable host animal.

The present invention also provides a model for study of humancardiomyocytes in culture, comprising the cardiomyocytes isolated by themethod of the current invention. This model may be used in thedevelopment of cardiomyocyte transplantation therapies.

According to yet another aspect the invention provides a method ofscreening for cardiovascular compounds. In particular the method maycomprise subjecting the said cardiomyocyte population to at least onetest compound, and observing a cardiac specific response of thecardiomyocytes to at least one test compound. In particular, thespecific cardiac response may be monitored by the changes of beatfrequency, amplitude and/or duration of the cardiomyocyte(s) to at leastone test compound. More in particular the cardiac specific response maycomprise alteration of Q-T wave.

There is also provided a kit for cardiotoxic testing or for screening ofcardiovascular compound(s) comprising at least one cardiomyocytepopulation according to the invention. There is also provided a kit forpreventing, repairing and/or treating at least one cardiac disorder in asubject, the kit comprising at least one cardiomyocyte populationaccording to the invention. The kit may further comprise instructionsfor use.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention.

EXAMPLES Materials and Methods hES Cell Culture

The hES cell line hES3 from ES Cell International(http://stemcells.nih.gov/research/registry/esci.asp) were maintained onhuman fibroblasts in KO-DMEM with 20% KOSR in 0.1 mMbeta-mercaptoethoethanal, 1% MEM non-essential amino acids, 2 mML-glutamine, bFGF (10 ng/ml) with or without antibiotics(Penicillin/Streptomycin; all reagents from Invitrogen). The hES cellswere passaged by treatment with collagenase I (however, collagenase IVmay also be used) (Gibco) for 3 minutes followed by mechanicaldissociation. Harvested cells were transferred to newly prepared feedercells.

hES Differentiation

The pluripotent hES grown on human feeders in 10 cm dishes were rinsedwith phosphate buffered saline (PBS). PBS was then replaced by freshstem cell maintenance medium. The dish was scored using a pipette tipsuch that each colony was divided approximately in two cell clusters.Cell clusters were scraped from the substrate and transferred to aconical tube. The cell clusters were allowed to settle to the bottom ofthe conical tube and the media was aspirated. The media was replaced byfresh stem cell maintenance medium. The cell clusters were transferredto plastic dishes to discourage cell attachment (Ultra-low attachdishes, Costar). The dishes were incubated in the tissue cultureincubators for a period of 24 hours. After the 24 hour culture, embryoidbodies (EBs) were formed from the pluripotent cell clusters insuspension. The dishes were tilted such that the embryoid bodies sank tothe bottom and the media was aspirated. The medium was replaced bydefined basic serum free (bSFS) medium comprising DMEM supplemented with1×MEM non-essential amino acids (Invitrogen), 2 mM L-Glutamine(Invitrogen), 0.0055 mg/ml Transferrin (Roche), 5 ng/ml sodium Selenite(Sigma), 0.1 mM beta-mercaptoethanol, with or withoutPenicillin/Streptomycin (Invitrogen). which promotes cardiomyocytedifferentiation as described in WO 2007/030870.

To further encourage differentiation towards the cardiomyocyte lineage,5 μg/ml of the compound SB203580, as described in WO 2007/030870, wasadded. The embryoid bodies were cultured in these conditions for anadditional 12 days. During this period, the culture medium was changedevery 3-4 days.

Digestion of Embryoid Bodies to a Single Cell Suspension.

The EBs were transferred to a conical tube and allowed to settle. Themedium was aspirated and the EBs rinsed with PBS not containing eithermagnesium or calcium. EBs were incubated at 37° C. in either undilutedAccumax reagent (Innovative Cell Technologies), or a 0.25 or 0.005%solution of trypsin (Roche) in phosphate buffered saline. The enzymaticreactions were arrested by the addition of differentiation mediumcontaining 20% fetal calf serum. Residual clusters of cells were removedby passing the cell suspension through a filter with maximum pore sizeof 40 μm.

Magnet Assisted Cells Sorting (MACS)

The single cell suspensions were pelleted in a centrifuge refrigeratedto 4° C. at approximately 300 gravities for 15 minutes. The cell pelletwas resuspended in an immunoglobulin blocking buffer (FcR blockingbuffer, Miltenyi Biotec) at a concentration of 1×10⁶ cells per 1000. Aconcentration of 0.5 to 5 μg/ml of antibody (mouse monoclonal ab23829,Abcam) which binds the cell surface antigen CD166 was added to the cellsuspension. The cell suspension was incubated for 30 minutes at 4° C.while rocking. The cells were pelleted again in a refrigeratedcentrifuge at approximately 300 gravities for 10 minutes. The blockingbuffer containing the anti-CD166 antibody was aspirated and replaced by80 ul per 1×10⁶ cells supplemented with 20 ul of magnetic microbeadconjugated antibody which recognizes the anti-CD166 antibody (ratanti-mouse igG2a+b microbeads, 472-01 Miltenyi Biotec) and was incubatedfor 30 minutes at 4° C. while rocking. The cells were pelleted andresuspended in fresh blocking buffer. Cells bound to magnetic microbeadswere separated from the unbound cell population by being passed througha column held in a strong magnetic field (Miltenyi Biotec columns,Miltenyi Biotec magnetic holder). The sorted cells were pelleted,resuspended in bSFS media containing 5 μM SB203580 and 20% fetal calfserum and plated in tissue culture dishes pre-coated with 100 μg/ml ofcollagen I (Roche). The media was changed every 2-3 days. After thecultures had grown to confluence, the medium was replaced by bSFS mediumcontaining 5 μM SB203580 but without fetal calf serum.

Quantitative PCR

Total RNA was isolated using the RNeasy kit (Qiagen), treated withon-filter DNase and quantified by UV absorption. One μg of RNA wasconverted to cDNA using M-MuLV reverse transcriptase (New EnglandBiolabs) using random hexamer primers and following manufacturer'sinstructions. Quantitative PCR was performed with 50 ng of each reversetranscriptase reaction, 250 nM of forward and reverse primer, 1×SYBRgreen PCR master mix (Bio-RAD) and analyzed by iCycler thermocycler(Bio-RAD). Primers comprising the sequence of SEQ ID NO:1 and SEQ IDNO:2 were used to detect binding amplification of the actin sequence,primers comprising the sequence of SEQ ID NO: 3 and SEQ ID NO:4 wereused to detect Nkx2.5, primers comprising the sequence of SEQ ID NO:5and SEQ ID NO:6 were used to detect αMHC sequence, primers comprisingthe sequence of SEQ ID NO: 7 and SEQ ID NO: 8 were used to NeuroD,primers comprising the sequence of SEQ ID NO. 9 and SEQ ID NO: 10 wereused to amplify oct4 sequence and primers comprising the sequence of SEQID NO. 11 and SEQ ID NO: 12 were used to amplify AFP sequence.Expression was calculated based on a standard curve and normalized to IIactin.

Immunofluorescence

The EBs were fixed in 4% paraformaldehyde, cryo-preserved in 25% sucroseat 4° C. overnight, snap frozen in OCT media (Leica), and sectioned to 6μm using a cryotome (Leica CM3050S). Sections were rinsed in PBS, fixedin 4% paraformaldehyde, permeabilized with 0.1% triton X-100 in PBS,incubated in block buffer (PBS, 0.1% Triton X-100, 1% BSA) and incubatedovernight at 4° C. in block buffer containing primary antibodies againstNkx2.5 (1:200 dilution, Santa Cruz), αMHC (1:100 dilution Santa Cruz),MLC2a (1:500 dilution, Chemicon), MLC2v (1:500 dilution, Chemicon),Tropomyosin (1:50 dilution, Iowa Developmental Studies Hybridoma Bank),or alpha-actinin (1:50 dilution, Chemicon). After three rinses in PBS,slides were incubated for one hour at room temperature in blockingbuffer containing secondary antibodies (1:1000 dilution, Chemicon,Zymed), incubated for one hour at room temperature, rinsed three timesin PBS, incubated in DAPI (1:2000 dilution) for 10 minutes, rinsed, andmounted with Fluorosave (Calbiochem).

BrdU Incorporation

Determination of cell proliferation was performed using in situ CellProliferation Kit, FLUOS (Roche) and following manufacturer'sinstructions. Briefly, 10 μM BrdU was added to the cell medium for aperiod of one or three hours. Cells were then fixed forimmunohistochemistry and DNA was denatured by 20 min incubation in 4MHCl.

Results Differentiation to Cardiomyocyte Lineage

Stem cells were stimulated to differentiate towards the cardiomyocytelineage following the methods described in WO 2007/030870. At the end ofthis culture period, lasting two weeks, clusters of cells in suspension,termed embryoid bodies (EBs) were produced. A large proportion of theEBs began spontaneous rhythmic contractions and contained cells whichexpressed markers of the cardiomyocyte lineage.

Differentiated EBs Contain Cells which Express the CardiomyocyteTranscription Factor Nkx2.5 and Cardiac Structural Proteins.

A highly specific and early marker of cardiac cell identity is thetranscription factor Nkx2.5. The Nkx2.5 marker is expressed ubiquitouslyin all mouse heart cell progenitors around the time the heart crescentis formed and is an important regulator of cardiac gene expression inthe developing and adult animals in both mice and humans (McFadden etal, 2002).

The marker Nkx2.5 was detected by immunofluorescence in cells of EBsdifferentiated according to the above protocol. Structural markers ofthe cardiac contractile machinery expressed in fetal cardiomyocytes wereco-expressed in cells expressing Nkx2.5, confirming their cardiacidentity (FIG. 1). It is known that αMHC and MLC2a are expressedthroughout the myocardium in the developing mouse heart (Somi at al,2006; Cai et al, 2005). FIG. 1A and FIG. 1B show that αMHC and MLC2awere co-expressed by clusters of cells which expressed Nkx2.5. Furthersince alpha actinin and tropomyosin are expressed in all cardiaccontractile tissue, the co-expression of these markers by cells whichexpressed Nkx2.5 was seen as shown in FIG. 1C and FIG. 1D. It is furtherknown that MLC2v expression is restricted to ventricle andatrioventricular canal when specification of these structures occurs(Cai et al, 2005). Accordingly MLC2v was not detected in differentiatedEBs, suggesting that these cells are homologous to a fetal developmentalstage wherein ventricular specification had not yet occurred (FIG. 1E).

Cardiomyocytes co-express surface markers useful for antibody-based cellselection.

The surface marker CD166 (ALCAM) is an adhesive molecule expressed inthe cardiac crescent and neural groove during mouse embryogenesis, andis lost in heart tissue by the time the mature heart has formed (Hirataet al, 2006). Therefore, cells isolated by expression of CD166 arelikely to be developmentally immature and have the capacity to replicatein culture. In this study CD166 was co-expressed with Nkx2.5 by cells inthe differentiated EBs, suggesting a fetal developmental stage of thesecells (FIG. 2A). The VEGF receptor Flk-1 is expressed by mouse cardiacprogenitors and is shown to be expressed in mouse embryonic stem cellswith potential to differentiate to beating cardiomyocytes (Moretti etal, 2006; Kattman et al, 2006). Accordingly Nkx2.5 expressing cells inthe EBs of the present invention was shown to co-express Flk-1 (FIG.2B). Further N-cadherin is expressed continuously during heartdevelopment, and is associated with cardiac progenitor cells isolatedfrom differentiating mouse embryonic stem cells (Honda et al, 2006).Accordingly Nkx2.5 expressing cells in the EBs also co-expressed thecell-cell adhesion molecule N-cadherin, (FIG. 2C).

Isolation of Cell Population Enriched for Cardiomyoctes

A single cell suspension prepared from differentiated EBs by gentledigestion with Accumax reagent was shown to survive better than thatwhen digested with trypsin, and better than undifferentiated humanembryonic stem cells digested by either method as shown in FIG. 3.Approximately 40% of differentiated cells digested using Accumax werecapable of adhering to a tissue culture dish and remaining viable for atleast 48 hours (FIG. 3). Subpopulations expressing the adhesion moleculeCD166 were isolated from single cell suspensions by MACS as described inthe materials and methods.

RNA extracted from cells immediately after sorting showed higherrelative quantities of Nkx2.5 and αMHC transcripts in the CD166expressing population than the CD166 negative or non-sorted populations(FIG. 4). In addition, cells sorted based on expression of CD166 havefewer transcripts of the neural marker NeuroD1 and the pluripotencymarker Oct4. Therefore the sorted cardiomyocyte population of thecurrent invention is depleted of non-cardiac cell types, includingresidual cells which presumably have the potential to form teratomasupon transplantation to a living animal.

Although only a small proportion of the starting differentiated cellpopulation may express CD166, one of the key features of the cellsisolated based on expression of CD166 is that the cells are capable ofreplication in culture. The sorted cells selected by this method havethe ability to grow rapidly in culture when plated at sub-confluentdensity. CD166− selected cells plated at approximately 30% confluence(FIG. 5A) in tissue culture dishes coated with collagen I in mediumcontaining 5-20% fetal calf serum were able to grow to 100% confluencein culture within 6 days (FIG. 5B). Further during this growth phase itwas seen 48 hours after plating that, approximately 25% of all adherentcells were in S-phase of the cell cycle as measured by BrdUincorporation (FIG. 5C). Alternatively, other adhesive substrates suchas fibronectin can be used to stimulate cardiomyocyte attachment to thetissue culture dish. In addition, the use of bovine serum can becircumvented by the addition of growth-stimulating factors in the mediumsuch as fibroblast growth factor or vascular endothelial growth factor.

It is important that the cells isolated by the method of the currentinvention retain their cardiac identity and have the potential to formfunctional, electrically coupled cardiomyocytes. It was seen thatpopulations of cells selected by expression of CD166 grown toconfluence, begin spontaneous contractions, implying the presence ofelectrically coupled, functional cardiomyocytes. When these cells werefixed and visualized for expression of the cardiac marker Nkx2.5 byimmunofluorescence, large clusters of cells expressed Nkx2.5. Visualcount of representative fields of Nkx2.5 expressing cells revealed thatcardiomyocytes represented greater than 50% of the total cell population(FIG. 6A). However cells that were negative for CD166 marker showed onlybasal level expression of Nkx2.5 (FIG. 6B). Cells expressing Nkx2.5 alsoco-expressed the cardiac structural marker myosin light chain 2a (MLC2a)confirming their cardiac identity as shown in FIG. 6C.

All the above experiments were performed with stringent controls. Theresults from the experiments suggest that identification and isolationof cardiomyocytes based on the expression of CD166 marker is anefficient way of obtaining a population of cells enriched incardiomyocytes. Further, in addition to the surface marker CD166,selection of cells can be based on expression of other markers includingFlk1, N-cadherin, CD133, and CD117. Further in addition to MACS, sortingof cells based on surface marker expression can be accomplished equallyas well using other methods known to those skilled in the art, forexample, FACS.

Finally, the invention as described herein is susceptible to variations,modifications and/or additions other than those specifically describedand it is understood that the invention includes all such variations,modifications and/or additions which fall within the scope of thedescription as described herein.

REFERENCES

-   Cai C L, Zhou W, Yang L, Bu L, Qyang Y, Zhang X, Li X, Rosenfeld M    G, Chen J, Evans S., 2005. T-box genes coordinate regional rates of    proliferation and regional specification during cardiogenesis.    Development. 132(10):2475-87.-   Hirata H, Murakami Y, Miyamoto Y, Tosaka M, Inoue K, Nagahashi A,    Jakt L M, Asahara T, Iwata H, Sawa Y, Kawamata S., 2006 ALCAM    (CD166) is a surface marker for early murine cardiomyocytes. Cells    Tissues Organs. 184(3-4):172-80.-   Honda M, Kurisaki A, Ohnuma K, Okochi H, Hamazaki T S, Asashima    M., 2006. N-cadherin is a useful marker for the progenitor of    cardiomyocytes differentiated from mouse ES cells in serum-free    condition. Biochem Biophys Res Commun. 351(4):877-82.-   Kattman S J, Huber T L, Keller G M., 2006. Multipotent flk-1+    cardiovascular progenitor cells give rise to the cardiomyocyte,    endothelial, and vascular smooth muscle lineages. Dev Cell.    11(5):723-32.-   McFadden D G, Olson E N., 2002. Heart development: learning from    mistakes. Curr Opin Genet Dev. 12(3):328-35. Review.-   Moretti A, Caron L, Nakano A, Lam J T, Bernshausen A, Chen Y, Qyang    Y, Bu L, Sasaki M, Martin-Puig S, Sun Y, Evans S M, Laugwitz K L,    Chien K R., 2006. Multipotent embryonic isl1+ progenitor cells lead    to cardiac, smooth muscle, and endothelial cell diversification.    Cell. 127(6):1151-65-   Siu C W, Moore J C, Li R A., 2007. Human embryonic stem cell-derived    cardiomyocytes for heart therapies. Cardiovasc Hematol Disord Drug    Targets. 7(2):145-52.-   Somi S, Klein A T, Houweling A C, Ruijter J M, Buffing A A, Moorman    A F, van den Hoff M J., 2006. Atrial and ventricular myosin    heavy-chain expression in the developing chicken heart: strengths    and limitations of non-radioactive in situ hybridization. J    Histochem Cytochem. 54(6):649-64.-   WO 2007/030870.

1. A method of identifying and selecting a cardiomyocyte population froma heterogeneous population of differentiated stem cells, comprisingcontacting the heterogeneous cell population with at least one agentthat specifically binds to at least one cardiomyocyte marker andselecting cells bound to the said agent as cardiomyocytes.
 2. The methodaccording to claim 1, further comprising a step of isolating theselected cardiomyocyte population.
 3. The method according to claim 2,further comprising a step of propagating the selected cardiomyocytepopulation in culture.
 4. The method according to claim 1, wherein thecardiomyocyte marker is selected from a group consisting of CD166(ALCAM), VEGF receptor Flk1, N-cadherin, CD133 and CD117 (C-kit).
 5. Themethod according to claim 1, wherein the cardiomyocyte marker is CD166(ALCAM).
 6. The method according to claim 1, wherein the cardiomyocytemarker is a fetal marker.
 7. The method according to claim 2, wherein atleast 50% of the isolated cells comprise cardiomyocytes.
 8. The methodaccording to claim 1, wherein the identified cardiomyocytes have a fetalphenotype.
 9. The method according to claim 1, wherein the identifiedcardiomyocytes are capable of proliferating in culture.
 10. The methodaccording to claim 1, wherein the identified cardiomyocytes are capableof rhythmic contractions and optionally form electrically coupled cellclusters.
 11. The method according to claim 1, wherein the stem cellsare selected from the group consisting of embryonic stem (ES) cell,pluripotent stem cells, hematopoietic stem cells, totipotent stem cells,mesenchymal stem cells, neural stem cells and adult stem cells.
 12. Themethod according to claim 11, wherein the stem cells are human ES cells.13. (canceled)
 14. A cardiomyocyte population, isolated by the methodaccording to claim
 2. 15. (canceled)
 16. A kit for cardiotoxic testingcomprising the cardiomyocyte(s) according to claim
 14. 17. A method ofpreventing, repairing or treating at least one cardiac disorder in asubject, the said method comprising transplanting the cardiomyocytepopulation isolated and/or enriched according to claim
 14. 18.(canceled)
 19. A method of screening for cardiovascular compounds usefulfor modulating cardiac cell functions, said method comprising contactinga cardiomyocyte population according to claim 14 with at least onecompound, and determining whether said compound produces a cardiacspecific response in the cardiomyocytes relative to untreatedcardiomyoctes.
 20. The method according to claim 19, wherein the cardiacspecific response comprises alteration of the Q-T wave.